Synthetic protein families powerfully complement natural genetic diversity in efforts to understand fundamental sequence-structure-function relationships. We propose to use a unique family of chimeric cytochrome P450s made by structure-guided recombination to make advances in modeling P450 structure, stability and specificity. Differing from one another by many dozens of amino acids yet still likely to fold into highly similar structures, these diverse sequences have different stabilities and exhibit an array of activities and specificities. We seek to maximize the number of accurate structure, stability, and specificity predictions given a set number of experimental measurements that are relatively easily performed on these laboratory-generated sequences. We propose to use this unique library of P450 chimeras to study enzyme structural modularity and exploit that modularity to simplify structure-function predictions. We will determine the X-ray crystal structures for several chimeric P450 heme domains. The crystal structures will assist our concurrent efforts to computationally predict accurate chimera structures. We will test new algorithms that recombine optimized enzyme substructures to sample relevant protein backbone conformations. Our goal is to bridge the gap between low resolution homology models and accurate high resolution structures. The chimeric enzymes are also a rich resource for investigating sequence-structure-stability relationships. The chimeric proteins have diverse stabilities;many are more stable than their parents. We plan to measure stability for selected chimeras and construct and test additive models for protein stability and quantify non- additive coupling effects. Recombination creates sequence intermediates between parent proteins and allows us to reconcile the effects of swapping blocks of sequence with the action of individual mutations. This work will contribute key data and insight into the effects of long-range interactions and coupling. Finally, we plan to explore the functional diversity of the chimeric cytochrome P450s, in the context of their applications in drug discovery and drug metabolism. These soluble, well-expressed chimeric P450s are a viable platform for production of authentic human metabolites of drugs and for lead diversification. Preliminary results indicate that the diverse sequences exhibit activity on a wide range of substrates. We will perform a systematic analysis of the activities of a set of chimeric P450s towards pharmaceutically relevant scaffolds, using clustering and regression to determine whether activity and specificity can be predicted from data on selected chimeras. We will attempt to rationalize the P450 specificity results in terms of structural P450- substrate complex models. This research continues an effective interdisciplinary collaboration that partners powerful experimental and theoretical capabilities. PUBLIC HEALTH RELEVANCE: We propose to demonstrate how a laboratory-generated family of cytochrome P450s can be used to study the basis of P450 structure, stability, and function. We will test a new strategy for predicting the interactions between drugs and P450 enzymes, a potential breakthrough that would allow us to predict drug metabolism and interactions and to efficiently produce drug metabolites for toxicity studies and drug discovery. Finally, this research represents a fundamentally new approach to study protein stability using recombination, with results that can be applied to understanding enzymes in general.